【作者】 张娜；

【导师】 韩家淮；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 TNF-α是一种重要的前炎症细胞因子,通过结合膜表面受体TNFRⅠ或TNFRⅡ,发挥其生物学功能,在众多细胞事件中发挥了重要作用。而只有结合带死亡结构域的TNFRⅠ,TNF-α才能诱导细胞死亡。依据具体处理条件和细胞对象的不同,TNF-α能诱导不同类型的死亡:凋亡或坏死。也就是说,TNF-α结合TNFRⅠ后,可以通过多种途径传递死亡信号。目前最为人知的是TNF-α通过TRADD、FADD、caspase-8介导的凋亡性死亡途径,众多caspase家族成员在这一事件中起了重要的作用。而在一些细胞中,TNF-α也能在caspases受抑制的情况下诱导细胞死亡,但如今人们还不十分了解这种死亡方式的信号转导机制。深入研究TNF-α诱导死亡的不同通路,了解它们的差别与关联,不仅可使我们进一步了解细胞死亡的调控机理,还有助于找出一些相关疑难病的治疗方法,为临床实践提供理论依据。本论文以两株NIH3T3细胞作为实验对象。它们在TNF-α刺激下分别通过不同途径传递死亡信号:NIH3T3(R)细胞借助caspases传递凋亡信号;而NIH3T3(S)细胞则通过另一条未知的,不依赖于caspases的信号通路执行死亡事件。由于这两株细胞最初来源是一致的,只是在长期的培养过程发生了自然突变,分化成两株在TNF-α刺激下有着不同表型的细胞,因此可以利用此二者同时研究TNF-α诱导下caspase依赖及非依赖的细胞死亡机制。为了初步探究两株细胞在TNF-α刺激下选择不同死亡途径的机理,我们利用cDNA表达谱芯片分析这两株细胞基因表达的差异,并通过逆转录PCR对差异基因进行验证。最终从众多的差异基因中选择Rip3作为本论文的研究对象,该基因仅在NIH3T3(S)细胞表达,而未在NIH3T3(R)细胞内表达。Rip3属于Rip激酶家族成员,拥有N末端Ser/Thr激酶活性区和独特的C末端,二者间有一个能介导同源相互作用的RHIM结构。现有研究已经表明与Rip3同家族成员Rip1是TNFRⅠ或Trif介导的NF-κB信号通路的重要成员,而Rip3会通过RHIM竞争结合并磷酸化Rip1,抑制NF-κB的激活。另外,有实验表明过量表达Rip3会诱导细胞凋亡,Rip3的C末端正是诱发凋亡的功能区。不过,总的来说,Rip3的功能研究尚处于探索阶段,仍有很多问题有待解决。本论文发现了Rip3的一个新功能:通过介导ROS积聚,传递TNF-α诱导的caspase非依赖的死亡信号。而这一过程的ROS的积聚需要有氧的糖酵解途径。这一发现将有助于进一步了解TNF-α诱导死亡的机制,在实践上则将提升TNF-α的临床应用价值。更多还原

【Abstract】 As a proinflammatory cytokine,TNF-αplays an important role in many cellular events.TNF-αelicits its biological effects by binding to the receptors,TNFRⅠand TNFRⅡ.But only TNFRⅠis a death receptor.By binding to the receptor TNFRⅠ, TNF-αinduces both apoptotic and necrotic cell death,depending on cell types and treatment condition.It means that TNF-αcan induce cell death through distinct signal transduction pathways.It has been extensively studied that the mechanisms of TNF-induced apoptotic cell death,which is mediated by TRADD,FADD and caspase-8.Caspases are required for apoptotic cell death.In some types of cells, TNF-αcan induced caspase-independent cell death,of which mechanism is still largely unknown.Further study elucidating the mechanism of TNF-induced cell death and difference and relationship among different signal pathways will facilitate our understanding of cell death control and be helpful for clinical treatment.In this study,we used two cell lines derived from NIH3T3 cells:NIH3T3(R) and NIH3T3(S).In NIH3T3(R) cell,TNF-αcan induce apoptosis which requires caspases; but in NIH3T3(S) cell,under a caspase-inhibited condition,TNF-αcan effectively induce cell death through an unknown signal pathway.We believe that these two cell lines would be good material for studying these two distinct mechanisms of the caspase-dependent or caspase-independent cell death induced by TNF-αat the same time,because which differentiated from the same ancestor in the long culture process. Large-scale gene expression profiles between these two cell lines were detected with cDNA microarray.As a result,one gene,namely Rip3,is selected for further study, which was detected only in NIH3T3(S) cell not in NIH3T3(R) cell in mRNA level, which was confirmed by RT-PCR.Rip3 is the member of the Rip family,which processes N-terminal Ser/Thr kinase domain,RHIM and a unique C-terminus.Rip1,belonging to the Rip family too,is an important NF-κB-activating molecule mediated by TNFR I or Trif.Rip3,via its RHIM,interacts with,and subsequently phosphorylates Rip1,resulting in diminished NF-κB activation.In addition,overexpression of Rip3 in some cell lines can induce apoptosis,which requires the C-terminus of Rip3.But additional studies are required to better understand the function of Rip3.This thesis reveals a novel role for Rip3,which is crucial in mediating ROS accumulation in TNF-induced caspase-independent cell death.And generation of cytocidal ROS requires an aerobic glycolytic pathway.This result will facilitate our understanding of the mechanism of TNF-induced cell death and improve the value in clinical application of TNF.更多还原